Antibody, nucleic acid, cell, and pharmaceutical

Abstract

To provide a novel anti-human CCR7 antibody that is useful as an antibody drug.SOLUTION: Provided is an antibody that specifically binds to an extracellular domain of human CCR7 and has a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, each containing a specified amino acid sequence. Preferably, the antibody has an Fc region, the Fc region contains an N-glycosidically linked sugar chain, fucose is not bound to the 6-position of N-acetylglucosamine at a reducing end of the N-glycosidically linked sugar chain, and the antibody has antibody-dependent cellular cytotoxicity.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present disclosure relates to an antibody that specifically binds to the extracellular domain of human CCR7, a nucleic acid encoding the antibody, a cell containing the nucleic acid, and a pharmaceutical comprising the antibody as an active ingredient. [Background technology]

[0002] Chemokines are proteins that regulate the migration and function of various cells. Dysfunction of chemokines and their receptors is a cause of various diseases, such as fibrosis, autoimmune diseases, acute and chronic inflammation, and cancer. Although drugs that control the activity of chemokines and their receptors have been developed and are being used clinically, it is difficult to say that they have fully resolved the problems.

[0003] Specific chemokines must bind to specific cell membrane receptors to exert their activities, such as regulating cell migration and cell function. Approximately 20 types of chemokine receptors have been discovered, and all of them are seven-transmembrane receptors (GPCRs) that bind to trimeric G proteins. When chemokines bind to their receptors, they release the Gα unit of the trimeric G protein, which then elevates intracellular Ca concentration and activates phosphatidylinositol 3-kinase (PI3K), small Rho GTPases, and other pathways, resulting in their functions. Although each chemokine receptor is activated by relatively specific chemokines, their primary protein structures and intracellular activation mechanisms are very similar. Therefore, it is not easy to selectively block the function of a specific chemokine receptor. The functional expression of each chemokine and chemokine receptor under physiological and pathological conditions is controlled by the expression of each protein in specific cells and tissues at specific times (during inflammation) (Non-Patent Document 1).

[0004] Human CC motif receptor 7 (CC MOTIF, RECEPTOR 7; also known as EBI1 or CMKBR7; hereafter referred to as "CCR7") was initially discovered as a GPCR selectively expressed in lymphocytes following Epstein-Barr virus infection (Non-Patent Document 2). CCR7 was subsequently found to be a selective chemokine receptor for CCL19 (also known as ELC) and CCL21 (also known as SLC or EXODUS 2). Under physiological conditions, CCR7 is relatively selectively expressed on CD4+ T cells (Th1, Th2, and Treg cells), mature dendritic cells, and B cells. It is known that these cells are recruited to inflammatory sites and other lesions via CCR7, enhancing inflammatory and immune responses. Abnormal CCR7 activation has also been implicated in various diseases, including autoimmune diseases, fibrosis following acute and chronic inflammation, and cancer metastasis.

[0005] In cancer treatment, it is important to suppress the growth of the primary cancer and prevent recurrence accompanied by distant metastasis. Although treatment outcomes have improved with conventional surgical therapy and chemotherapy, as well as molecularly targeted drugs (e.g., kinase inhibitors), antibody drug therapy, and cancer immunotherapy, there is still a need for the development of therapeutic agents that prevent recurrence accompanied by distant metastasis. Distant metastasis can occur via blood vessels from the primary cancer or via lymphatic tissue. To date, inhibitors of extracellular matrix proteinases (MMP inhibitors) have been developed as drugs to prevent metastasis, but no clinical applications have been achieved.

[0006] Various studies have shown that CCR7 is expressed in various tumor cells, such as B-cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer cells, and malignant breast tumors. Furthermore, it has become clear that CCR7 plays a role in lymph node metastasis of various cancers, such as gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, T-cell leukemia, cervical cancer, various squamous cell carcinomas, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma (Non-Patent Document 1). Because CCR7 ligands CCL19 and CCL21 are highly expressed in lymph nodes, selective inhibition of CCR7 function is expected to suppress lymphatic metastasis of cancer cells.

[0007] Potential candidates for selective CCR7 function inhibitors include small molecule compounds and monoclonal antibodies that selectively bind to the extracellular domain of the receptor and block the CCR7 signal transduction mechanism induced by CCL19 / CCL21 stimulation.

[0008] Patent Document 1 discloses eight monoclonal antibodies that specifically bind to the extracellular domain of human CCR7. It also discloses the amino acid sequences of the complementarity determining region (CDR), heavy chain variable region, and light chain variable region of each monoclonal antibody. These anti-human CCR7 antibodies are considered to be particularly effective in treating fibrosis.

[0009] Patent Document 2 discloses a humanized version of one type of monoclonal antibody that specifically binds to human CCR7, and discloses that the humanized antibody or an Fc variant with enhanced complement-dependent cytotoxicity kills multiple tumor cells of blood cancers via its effector function.

[0010] Patent Document 3 discloses an antibody-drug conjugate in which an anticancer drug is bound to a monoclonal antibody that specifically binds to human CCR7 and is a mutant that has no effector function.

[0011] However, the antibodies described in Patent Documents 1 to 3 have not yet been put to practical use as therapeutic agents. There is a need in the art for alternative or improved anti-human CCR7 antibodies with superior properties as pharmaceutical raw materials.

[0012] The properties required for monoclonal antibodies as raw materials for antibody drugs that inhibit CCR7 function include high receptor selectivity, strong functional inhibitory activity, high solubility, thermal stability, low aggregation, and low immunogenicity. All of these are necessary properties for the practical application of antibody drugs, but low immunogenicity in humans is particularly important in terms of the safety of antibody drugs and determining the administration period. The importance of low immunogenicity is also indicated in the guidelines for the development of biopharmaceuticals from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA).

[0013] A common method for achieving low immunogenicity of antibodies is antibody humanization. Specifically, in order to reduce immunogenicity while maintaining high receptor specificity and strong functional inhibitory activity, antibodies obtained using rodents can be humanized using CDR grafting while maintaining 100% of the amino acid sequences of heavy chain CDRs 1-3 and light chain CDRs 1-3. However, there are only two types of antibodies against GPCRs on the market as pharmaceuticals, and it is not easy to reduce immunogenicity while maintaining high target molecule specificity and strong functional inhibitory activity.

[0014] In addition to direct blockade of the receptor's intracellular signaling pathway, antibody therapeutics are known to exert antitumor activity in vivo through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC). ADCC occurs when an antibody binds to an antigen on a cancer or target cell, attracting immune cells such as macrophages and natural killer (NK) cells with Fc receptors that recognize the antibody's Fc region to the target cell, resulting in the killing of the antibody-bound cancer or target cell. Antibodies with ADCC activity, in addition to neutralizing activity, are considered useful in cancer therapy because they exert antitumor activity at low doses. The strength of ADCC activity is determined by the amount of antigen expression on the target cell, the strength of antigen-antibody binding, antibody selectivity, and the affinity of the antibody's Fc region with the Fc receptor. It is not clear whether all GPCR antibodies possess ADCC activity; the strength of ADCC activity varies from antibody to antibody.

[0015] Methods for artificially enhancing the ADCC activity of antibodies include modifying the amino acid sequence of the antibody's Fc region and controlling the antibody's glycan structure, which is known to be controlled by removing fucose from the reducing end of the antibody's N-type complex glycan (defucosylation). [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2012 / 043533 [Patent Document 2] International Publication No. 2017 / 025569 [Patent Document 3] International Publication No. 2021 / 220199 [Non-patent literature]

[0017] [Non-Patent Document 1] Viola A, Luster AD "Chemokines and their receptors: drug targets in immunity and inflammation", Annu Rev Pharmacol Toxicol. 2008;48:171-197 [Non-patent document 2] Birkenbach, M., Josefsen, K., Yalamanchili, R., Lenoir, G., Kieff, E., "Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors", J. Virol. 67: 2209-2220, 1993. Summary of the Invention [Problem to be solved by the invention]

[0018] As described above, antibodies that inhibit the function of CCR7 are expected to be useful as antibody drugs, but none have yet been put to practical use as therapeutic agents. Therefore, the present disclosure aims to provide a novel anti-human CCR7 antibody with superior properties as a pharmaceutical ingredient. [Means for solving the problem]

[0019] The present inventors humanized a monoclonal antibody that blocks the CCR7 signaling mechanism, as described in Patent Document 1, using a CDR grafting method. However, this method failed to produce an antibody with the desired low immunogenicity for pharmaceutical use. Therefore, the inventors modified the amino acid sequences of the variable regions, including the CDRs, of known monoclonal antibodies that block the CCR7 signaling mechanism and examined their functional inhibitory activity and immunogenicity. As a result, they succeeded in obtaining a novel anti-human CCR7 antibody that has a CDR amino acid sequence different from that of existing antibodies and has low immunogenicity. Furthermore, they found that this antibody has cytocidal activity through ADCC activity against cancer cells, particularly leukemia cells that highly express CCR7. Furthermore, they unexpectedly succeeded in enhancing ADCC activity by defucosylation of the antibody's glycosylation.

[0020] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 27, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 29, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39.

[0021] Preferably, the antibody has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:31.

[0022] Preferably, the antibody has an Fc region, the Fc region contains an N-glycoside-linked sugar chain, fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain, and the antibody has antibody-dependent cellular cytotoxicity.

[0023] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 21 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 21, and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 31 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 31; and has an Fc region, wherein the Fc region comprises an N-glycoside-linked glycan in which fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked glycan; and has antibody-dependent cellular cytotoxicity.

[0024] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 45, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 49, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 59.

[0025] Preferably, the antibody has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:41 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:51.

[0026] Preferably, the antibody has an Fc region, the Fc region contains an N-glycoside-linked sugar chain, fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain, and the antibody has antibody-dependent cellular cytotoxicity.

[0027] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 41 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 41, and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 51 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 51; and an Fc region comprising an N-glycoside-linked sugar chain in which fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain; and has antibody-dependent cellular cytotoxicity.

[0028] A nucleic acid according to one embodiment of the present disclosure encodes the above-described antibody.

[0029] Preferably, the nucleic acid comprises a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO:21 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:31.

[0030] Preferably, the nucleic acid comprises a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO:41 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:51.

[0031] A cell according to one embodiment of the present disclosure comprises the nucleic acid described above.

[0032] A pharmaceutical according to one aspect of the present disclosure contains the above-described antibody as an active ingredient.

[0033] Preferably, the medicament is used for the treatment of cancer.

[0034] Preferably, the cancer is a blood cancer.

[0035] Preferably, the blood cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or non-Hodgkin's lymphoma.

[0036] Preferably, the non-Hodgkin's lymphoma is B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, chronic lymphocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, mature T / NK cell derived, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, or cutaneous lymphoma.

[0037] Preferably, the cancer is a solid cancer.

[0038] Preferably, the solid cancer is breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, cervical cancer, hepatocellular carcinoma, urothelial carcinoma, renal cell carcinoma, or squamous cell carcinoma.

[0039] Preferably, the squamous cell carcinoma is oral squamous cell carcinoma, esophageal squamous cell carcinoma, or pharyngeal squamous cell carcinoma. [Effects of the Invention]

[0040] According to the present disclosure, it is possible to provide a novel anti-human CCR7 antibody that has superior properties as a pharmaceutical raw material. [Brief explanation of the drawings]

[0041] [Figure 1A] FIG. 1 is an explanatory diagram showing the alignment of the heavy chain variable regions of multiple humanized anti-CCR7 antibodies obtained in the Examples. [Figure 1B] FIG. 1 is an explanatory diagram showing the alignment of the light chain variable regions of multiple humanized anti-CCR7 antibodies obtained in the Examples. [Figure 2] 1 is a graph showing the relationship between the inhibitory activity of an antibody against intracellular Ca 2+ signals and the antibody concentration. [Figure 3] 1 is a graph showing the number of immunogenicity-positive donors for each antibody. [Figure 4A]1 is a graph showing the relationship between antibody concentration and fluorescence intensity of a flow cytometry histogram in hCCR7 gene-transfected cells. [Figure 4B] 1 is a graph showing the relationship between antibody concentration and fluorescence intensity of flow cytometry histograms in Granta-519 cells. [Figure 4C] 1 is a graph showing the relationship between antibody concentration and fluorescence intensity of flow cytometry histograms in MJ cells. [Figure 5A] 1 is a graph showing the results of evaluating the inhibitory activity of NB007-01 on intracellular Ca 2+ signals in hCCR7 gene-transfected cells stimulated with CCL21. [Figure 5B] 1 is a graph showing the results of evaluating the inhibitory activity of NB007-01 on intracellular Ca 2+ signals in hCCR7 gene-transfected cells stimulated with CCL19. [Figure 6A] 1 is a graph showing the results of evaluating the inhibitory activity of NB007-01 on intracellular Ca 2+ signals in MJ cells stimulated with CCL21. [Figure 6B] 1 is a graph showing the results of evaluating the inhibitory activity of CAP-100 on intracellular Ca 2+ signals in MJ cells stimulated with CCL21. [Figure 7] 1 is a graph showing the results of evaluating the inhibitory activity of NB007-01 against CCL19-induced cell migration in Granta-519 cells. [Figure 8A] 1 shows an HPLC chromatogram analyzing the glycan structure of defucosylated NB007-01. [Figure 8B] FIG. 8B is an explanatory diagram showing the abundance ratio of each sugar chain species calculated from the chromatogram of FIG. 8A. [Figure 9] 1 shows graphs showing the results of evaluating the cytotoxic function of each antibody using the ADCC Reporter Bioassay method. [Figure 10] 1 shows graphs depicting the results of evaluating the cytotoxic function of each antibody by a cytotoxic activity assay using human PBMCs. [Figure 11]1 is a graph showing the results of evaluating the antitumor effect of antibodies using a Granta-519 cell transplant model. [Figure 12A] 1 is a graph showing the results of evaluating the inhibitory effect of antibodies on cell infiltration into lymph nodes using a Granta-519 cell transplant model. [Figure 12B] Fig. 10 is a graph showing the results of evaluating the inhibitory effect of antibodies on cell infiltration into the liver using a Granta-519 cell transplant model. DETAILED DESCRIPTION OF THE INVENTION

[0042] In the present disclosure, complementarity determining region is abbreviated as CDR. In the present disclosure, the heavy chain variable region may be abbreviated as VH, the heavy chain constant region as CH, the light chain variable region as VL, and the light chain constant region as CL. In the present disclosure, the term "antibody" may be replaced with "immunoglobulin." In the present disclosure, the term "nucleic acid" may be replaced with "DNA" or "gene."

[0043] In the present disclosure, a humanized antibody refers to an antibody in which the CDRs are derived from a non-human animal and the other regions (framework regions, constant regions, etc.) are derived from humans. In the present disclosure, a chimeric antibody refers to an antibody in which the heavy chain variable region (VH) and light chain variable region (VL) are derived from a non-human animal and the other regions, such as the heavy chain constant region (CH) and light chain constant region (CL), are derived from humans.

[0044] <Human CCR7> CCR7 is a type of G protein-coupled receptor (GPCR) that penetrates the cell membrane seven times and is present with its N-terminus facing extracellularly and its C-terminus facing intracellularly. The gene (cDNA) encoding human CCR7 has already been isolated, and the amino acid sequence of human CCR7 is also known. This sequence information can be obtained, for example, from databases such as GenBank (e.g., GenBank: EAW60669.1). As an example, SEQ ID NO: 81 shows the nucleotide sequence of the human CCR7 gene. SEQ ID NO: 82 shows the amino acid sequence encoded by this nucleotide sequence.

[0045] Each domain of human CCR7 is thought to correspond to the following portion of the amino acid sequence shown in SEQ ID NO: 82. The left side indicates the amino acid number, and the right side indicates each domain. Note that the boundaries between each domain may vary slightly.

[0046] 1-24: Membrane translocation signal peptide sequence (cleaved and removed after expression) 25-59: N-terminal domain 87-95: Intracellular first loop domain 117-130: Extracellular first loop domain 153-170: Intracellular second loop domain 192-219: extracellular second loop domain 248-263: Intracellular third loop domain 290-313: extracellular third loop domain 332-378: C-terminal domain

[0047] Various variants of human CCR7, such as amino acid substitutions, are known in addition to the variant shown in SEQ ID NO: 82. In the present disclosure, "human CCR7" includes such variants as long as they have an extracellular domain and the function of CCR7.

[0048] <Anti-CCR7 antibody> The antibody disclosed herein specifically binds to the extracellular domain of human CCR7. The antibody according to one embodiment has a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39. Preferably, the antibody is a humanized antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31. An example of the antibody according to this embodiment is NB007-01, which is described in the Examples below.

[0049] The heavy chain variable region (SEQ ID NO: 21) comprises the heavy chain CDRs 1 to 3 (SEQ ID NOs: 25, 27, and 29), with the regions other than the CDRs derived from a human antibody. Similarly, the light chain variable region (SEQ ID NO: 31) comprises the light chain CDRs 1 to 3 (SEQ ID NOs: 35, 37, and 39), with the regions other than the CDRs derived from a human antibody.

[0050] An antibody according to another embodiment has a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 45, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59. Preferably, the antibody is a humanized antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51. An example of an antibody according to this embodiment is NB007-02, which is described in the Examples below.

[0051] The heavy chain variable region (SEQ ID NO: 41) comprises the heavy chain CDRs 1 to 3 (SEQ ID NOs: 45, 47, and 49), with the regions other than the CDRs derived from a human antibody. Similarly, the light chain variable region (SEQ ID NO: 51) comprises the light chain CDRs 1 to 3 (SEQ ID NOs: 55, 57, and 59), with the regions other than the CDRs derived from a human antibody.

[0052] Furthermore, the present disclosure includes an antibody that specifically binds to the extracellular domain of human CCR7, which has a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19. Preferably, the antibody is a chimeric antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11. Examples of such antibodies include VH0 / VL0 described in the Examples below.

[0053] Furthermore, the present disclosure includes an antibody that specifically binds to the extracellular domain of human CCR7, which has a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69, a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 79. Preferably, the antibody is a humanized antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 71. An example of such an antibody is NB007-03, which is described in the Examples below.

[0054] The antibody may be a functional fragment of an antibody. Here, "functional fragment of an antibody" refers to a partial fragment of an antibody (i.e., immunoglobulin) that retains at least one function against an antigen. Examples of such partial fragments include F(ab')2, Fab, Fv, disulfide-linked Fv, single-chain antibodies (scFv, VH-VL), etc. Furthermore, the antibody of the present disclosure may be a multispecific antibody such as a diabody.

[0055] When the antibody is a functional fragment, it has the following effects, for example. Specifically, when applying the anti-human CCR7 antibody of the present disclosure to pharmaceuticals as described below, using a full-length antibody such as an IgG type antibody may not only inhibit the signaling of the target receptor but also cause damage to the target tissue, which may lead to side effects. In such cases, using a "functional antibody fragment" using only the variable region makes it easier to avoid the side effects described above.

[0056] The above-mentioned antibody preferably has the activity of blocking a CCR7-dependent intracellular signal transduction mechanism induced by CCR7 ligand stimulation.

[0057] The antibody preferably has antibody-dependent cellular cytotoxicity (ADCC) activity, which makes the antibody particularly suitable as an active ingredient in cancer therapeutic drugs.

[0058] The present disclosure includes antibodies that are "functionally equivalent" to the above-described anti-human CCR7 antibodies. For example, the present disclosure discloses antibodies that specifically bind to the extracellular domain of human CCR7, and have a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 21 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 21, and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 31 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 31, and that have antibody-dependent cellular cytotoxicity.

[0059] Similarly, an antibody that specifically binds to the extracellular domain of human CCR7, which comprises an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 41, or comprises an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 41, a heavy chain variable region, and an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 51, or comprises an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 51, a light chain variable region, and having antibody-dependent cell cytotoxic activity is disclosed.

[0060] The number of the above-described substituted, added, or deleted amino acids is preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3. The identity of the above-described amino acid sequences is preferably 92% or more, more preferably 95% or more, and particularly preferably 97% or more.

[0061] <Defucosylation of the Fc region> The above anti-human CCR7 antibody has an Fc region, the Fc region contains an N-glycoside-linked sugar chain, and it is preferable that fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain. Briefly speaking, it is preferable that the N-glycoside-linked sugar chain of the Fc region is defucosylated. More specifically speaking, it is preferable that core fucose is not bound to the N-glycoside-linked sugar chain of the Fc region. By defucosylating the N-glycoside-linked sugar chain of the Fc region, it can be expected that the ADCC activity of the antibody increases and the immunogenicity of the antibody decreases.

[0062] Antibodies in which the N-glycoside-linked sugar chains of the Fc region have been defucosylated (defucosylated antibodies) can be produced, for example, by using host cells in which the activity of enzymes involved in fucose synthesis is reduced or deleted. Examples of such enzymes include GDP-mannose 4,6-dehydratase (GMD), GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase (Fx), GDP-beta-L-fucose pyrophosphorylase (GFPP), and alpha-1,6-fucosyltransferase (FUT8). Examples of such host cells include CHO cells.

[0063] When actually producing a defucosylated antibody, it may be obtained as an antibody composition consisting of a mixture of a defucosylated antibody and a non-defucosylated antibody. The molar ratio of the defucosylated antibody to the non-defucosylated antibody in the antibody composition is not particularly limited, but preferably the defucosylated antibody accounts for 50% or more, more preferably 70% or more, and particularly preferably 80% or more. In other words, of the N-glycoside-linked sugar chains contained in the antibody composition, preferably 50% or more, more preferably 70% or more, and particularly preferably 80% or more are defucosylated.

[0064] <Nucleic acid> The present disclosure includes nucleic acids (DNA) encoding the above-described antibodies. The nucleic acids include, for example, a first nucleic acid encoding a heavy chain variable region and / or a second nucleic acid encoding a light chain variable region. Specific examples include those containing a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 21 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 31. Other examples include those containing a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 41 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 51.

[0065] The nucleic acid may be incorporated into a vector. The vector is appropriately selected depending on the type of host cell to be introduced, etc. Vectors include vectors for gene therapy.

[0066] <cell> The present disclosure encompasses cells containing the nucleic acid. For example, a cell containing a vector incorporating the nucleic acid is an example of such a cell. The type of cell is not particularly limited as long as it is capable of expressing the nucleic acid, for example, in which the vector functions. Examples include animal cells (COS cells, CHO cells, etc.), yeast, bacteria (Escherichia coli, etc.), plant cells, insect cells, etc.

[0067] <Antibody manufacturing method> The antibody can be produced using genetic recombination techniques, i.e., by constructing recombinant cells that express the nucleic acid, the antibody can be obtained from a culture of the cells.

[0068] An example of a method for constructing and producing a humanized antibody is given below. First, DNAs encoding the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 are prepared, each encoding the amino acid sequences shown in SEQ ID NOs: 25, 27, 29, 35, 37, and 39. Examples of such DNAs include the nucleotide sequences shown in SEQ ID NOs: 26, 28, 30, 36, 38, and 40, although other nucleotide sequences may also be used.

[0069] In another example, DNAs encoding heavy chain CDR1 to 3 and light chain CDR1 to 3 are prepared by encoding the amino acid sequences shown in SEQ ID NOs: 45, 47, 49, 55, 57, and 59. Examples of such DNAs include the nucleotide sequences shown in SEQ ID NOs: 46, 48, 50, 56, 58, and 60, but other nucleotide sequences may also be used.

[0070] Next, these DNAs are used to prepare DNAs encoding variable regions in which heavy chain CDRs 1 to 3 are grafted into the framework regions (FR) of VH of any human antibody. Similarly, DNAs encoding variable regions in which light chain CDRs 1 to 3 are grafted into the FR of VL of any human antibody are prepared. Each of the prepared DNAs is inserted into a vector containing a sequence encoding the CH or CL of a human antibody to construct a humanized antibody expression vector. The constructed expression vector is introduced into host cells to obtain recombinant cells that express the humanized antibody. The recombinant cells are then cultured, and the desired humanized antibody is obtained from the culture.

[0071] The method for purifying the antibody is not particularly limited, and any known method can be used. For example, the culture supernatant of the recombinant cell can be collected, and the antibody can be purified by a combination of known methods such as various types of chromatography, salting out, dialysis, and membrane separation.

[0072] <Pharmaceuticals> The present disclosure encompasses a pharmaceutical comprising the anti-human CCR7 antibody as an active ingredient. The pharmaceutical may be a pharmaceutical composition comprising the anti-human CCR7 antibody and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical blocks CCR7-dependent intracellular signal transduction mechanisms induced by CCR7 ligand stimulation. Preferably, the pharmaceutical has antibody-dependent cellular cytotoxicity (ADCC) activity.

[0073] In a preferred embodiment, the medicament is used to treat cancer. For example, the medicament is used as an anticancer agent. The cancer may be either a blood cancer or a solid cancer.

[0074] Examples of blood cancers include acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, etc. Examples of non-Hodgkin's lymphomas include B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, chronic lymphocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, mature T / NK cell-derived, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, cutaneous lymphoma, etc.

[0075] Examples of solid cancers include breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, cervical cancer, hepatocellular carcinoma, urothelial carcinoma, renal cell carcinoma, squamous cell carcinoma, etc. Examples of squamous cell carcinomas include oral squamous cell carcinoma, esophageal squamous cell carcinoma, pharyngeal squamous cell carcinoma, etc.

[0076] Here, the term "treatment" means preventing or alleviating the progression and worsening of the pathological condition of a disease in a mammal that is at risk of contracting or is contracting the disease, and is used to mean a therapeutic procedure aimed at preventing or alleviating the progression and worsening of the symptoms of the disease.

[0077] <Administration method> The above-mentioned pharmaceuticals can be administered orally or parenterally, systemically or locally. Examples of administration forms include injections, intranasal administration, pulmonary administration, and transdermal administration. Injections can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. The administration method can be selected appropriately depending on the age and symptoms of the patient. The dosage of the above-mentioned antibody can be selected, for example, from a range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from a range of 0.001 to 100,000 mg of antibody per patient. However, the dosage of the above-mentioned antibody is not limited to these ranges.

[0078] <Formulation> The above-mentioned pharmaceuticals can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). The above-mentioned pharmaceuticals can contain pharmaceutically acceptable carriers and additives. Examples of the carriers and additives include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphate, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents, but are not limited to these. Other commonly used carriers and the like can also be used as appropriate. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. Furthermore, the composition may contain other low-molecular-weight polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, etc., and amino acids such as glycine, glutamine, asparagine, arginine, lysine, etc.

[0079] When preparing an aqueous solution for injection, examples include physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and these may be used in combination with appropriate solubilizing agents, such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. If necessary, the antibody can also be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.) or made into colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.) (see, for example, "Remingto's Pharmaceutical Science 16th edition", Oslo Ed. 1980)).

[0080] Furthermore, techniques for sustained release of drugs are known and can be applied to the above-mentioned pharmaceuticals (Langer et al., J. Biomed. Master. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:9(8-105 (1982); U.S. Pat. No. 3,773,919; European Patent Application Publication No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); European Patent Application Publication No. 133,988).

[0081] The above-mentioned drugs can be administered as antibody-drug conjugates (ADCs), which can be combined with antitumor drugs such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0082] <Application to gene therapy> The nucleic acid can also be incorporated into a gene therapy vector to produce a gene therapy drug. Methods for administering the gene therapy drug (recombinant vector) include direct administration using a naked plasmid, administration by packaging the vector in liposomes, administration by incorporating the vector into various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, and HVJ vectors (see Adolph, "Viral Genome Methods," CRC Press, Florida (1996)), and administration by coating the vector onto a bead carrier such as colloidal gold particles (e.g., WO 93 / 17706).

[0083] The gene therapy drug may be administered by any method as long as the antibody is expressed in vivo and can exert its effect. Preferably, a sufficient amount is administered via an appropriate parenteral route, such as intravenous, intraperitoneal, subcutaneous, intradermal, intraadipose tissue, intramammary tissue, inhalation, or intramuscular route, including injection, infusion, gas-induced particle bombardment (using an electron gun, etc.), or mucosal administration such as nasal spray. Furthermore, the gene therapy drug may be administered ex vivo to cells using liposome transfection, particle bombardment (U.S. Pat. No. 4,945,050), or viral infection, and then the cells may be reintroduced into an animal.

[0084] <Other Disclosures> The present disclosure includes a method of treating cancer comprising administering to a cancer patient an effective amount of the antibody. The present disclosure includes the antibody for use in cancer treatment. The present disclosure includes use of the antibody for manufacturing a medicament for use in cancer treatment. [Example]

[0085] Example 1: Preparation of humanized anti-CCR7 antibody To create a humanized version of the murine anti-CCR7 antibody R7-18 (described in International Publication No. 2012 / 043533 and Japanese Patent No. 5315495), we first constructed a chimeric anti-CCR7 antibody (VH0 / VL0) by fusing the heavy and light chain variants of R7-18 with the IgG1 Fc region of a human antibody. Furthermore, human frameworks were selected based on the homology between R7-18 and human germline VH and VK genes. Based on computer modeling, we designed multiple variable region sequences by adding mutations to the selected human germline VH and VK or grafted CDR sequences of R7-18 to support the predicted antibody conformation of R7-18. Among the designed humanized antibodies, "VH5-2 / VL1-3," "VH5-1 / VL1," and "VH5 / VL5," which maintained CCR7 neutralizing activity, were selected and named "NB007-01," "NB007-02," and "NB007-03," respectively. NB007-01, NB007-02, NB007-03, and the reference antibody CAP-100 (described in Patent Document 2) were produced using the QMCF method (described in International Publication No. 2006 / 084754 and Published Japanese Translation of PCT International Publication No. 2008-529510).

[0086] Alignment of the heavy chain variable region VH0 (SEQ ID NO: 1) of VH0 / VL0, the heavy chain variable region VH5-2 (SEQ ID NO: 21) of designed NB007-01, the heavy chain variable region VH5-1 (SEQ ID NO: 41) of designed NB007-02, and the heavy chain variable region VH5 (SEQ ID NO: 61) of designed NB007-03 is shown in Figure 1A. Alignment of the light chain variable region VL0 (SEQ ID NO: 11) of VH0 / VL0, the light chain variable region VL1-3 (SEQ ID NO: 31) of designed NB007-01, the light chain variable region VL1 (SEQ ID NO: 51) of designed NB007-02, and the light chain variable region VL5 (SEQ ID NO: 71) of designed NB007-03 is shown in Figure 1B. In Figures 1A and 1B, the regions covering all of the CDRs defined by the KABAT and IMGT methods are enclosed in dashed boxes.

[0087] The amino acid sequences (AA) of the heavy chain variable region, heavy chain constant region, heavy chain CDR1 to 3, light chain variable region, light chain constant region, and light chain CDR1 to 3 of each antibody obtained, as well as the nucleotide sequences of the DNA encoding them, are summarized in Tables 1-1 to 1-8.

[0088] [Table 1-1]

[0089] [Table 1-2]

[0090] [Table 1-3]

[0091] [Table 1-4]

[0092] [Table 1-5]

[0093] [Table 1-6]

[0094] [Table 1-7]

[0095] [Table 1-8]

[0096] Example 2: Efficacy of humanized anti-CCR7 antibodies Human CCR7 gene-transfected cells (described in International Publication No. 2012 / 043533 and Japanese Patent No. 5315495) were placed in a 96-well microplate at 2 × 10 cells per well. 4 The cells were seeded at an initial cell density of 100 μL and cultured for 2 days. After 2 days, the culture medium was replaced with a solution containing 3 μM Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Invitrogen). After 1 hour, 10 cells were added to each well. -6 ~10 -10 The humanized anti-CCR7 antibodies NB007-01, NB007-02, or NB007-03 were added at a concentration range of 10 M. As a positive control, the chimeric antibody VH0 / VL0 was added at a concentration of 10 M. -6 ~10 -10 It was added within the concentration range of M.

[0097] After 15 minutes, 5 x 10 -8 Each cell was stimulated with CCL21 (R&D Systems) containing M. 2+ Measurement of Ca concentration 2+ The signal was measured using a signal measurement system (FDSS / μCELL; Hamamatsu Photonics) to measure the intracellular Ca concentration of each additive (antibody). 2+ The inhibitory activity of the antibody against intracellular Ca signaling was analyzed. The results are shown in Figure 2 and Table 2. Figure 2 shows the 2+ This is a graph showing the relationship between inhibitory activity against signals and antibody concentration. Inhibitory activity was calculated as a relative value by standardizing the inhibition rate of "no antibody but with ligand" to 0% and the inhibition rate of "no antibody and no ligand" to 100%. Table 2 shows the 50% inhibitory concentration (IC50) of each antibody calculated from the analysis results of Figure 2.

[0098] As shown in Figure 2, VH0 / VL0, NB007-01, NB007-02, and NB007-03 all increased intracellular Ca in a concentration-dependent manner. 2+ This is because each additive inhibited the binding of CCL21 to human CCR7, resulting in an increase in intracellular Ca 2+This indicates that signal transduction was inhibited. The IC50 values ​​were 16.2 nM for VH0 / VL0, 18.2 nM for NB007-01, 16.4 nM for NB007-02, and 12.5 nM for NB007-03 (Table 2). These results demonstrate that the obtained humanized anti-CCR7 antibodies all have CCR7 inhibitory activity equivalent to that of the chimeric antibody VH0 / VL0.

[0099] [Table 2]

[0100] [Example 3] Immunogenicity of humanized anti-CCR7 antibodies The immunogenicity of NB007-01, NB007-02, and NB007-03 against human PBMCs was evaluated.

[0101] (1) Preparation of human PBMCs Human PBMCs were isolated from whole blood collected from healthy volunteers using a density gradient method, and then cryopreserved in human AB serum or fetal bovine serum with 10% dimethyl sulfoxide. Human PBMCs were stored at -180°C until use.

[0102] (2) DC-T cell assay Monocytes were isolated from human PBMCs by magnetic separation and cultured for 5 days in DC medium containing interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to differentiate into immature DCs (iDCs). iDCs were harvested, seeded onto cell culture plates, and differentiated into mature DCs by adding the test compounds NB007-01, NB007-02, or NB007-03. Then, they were cultured overnight in DC medium containing interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α). NB007-01, NB007-02, and NB007-03 and the cytokine cocktail were removed by washing.

[0103] CD4+ T cells were isolated from human PBMCs using magnetic separation with negative selection (StemCells: EasySep TM CD4+ T cells were isolated using the Human CD4+ T Cell Enrichment Kit (19052). CD4+ T cells and mature DCs were cocultured in serum-supplemented medium for 6 days. 5-Ethynyl-2'-deoxyuridine (EdU) was added to the coculture of mature DCs and T cells. After 16 hours, live and dead cells were separated by fluorescent labeling and further stained for T cell surface markers CD3 and CD4. After fixation and permeabilization, EdU incorporation was stained with fluorescent azide and analyzed by flow cytometry (LSR Fortessa, BD). Proliferating Th cells were defined as CD3-, CD4-, and EdU-positive cells and analyzed using FlowLogic software.

[0104] (3) Statistical analysis The positive response of the donors to the test substance (NB007-01, NB007-02, or NB007-03) was evaluated by calculating the stimulation index (SI), defined as the ratio of the mean response of the test substance to the mean response of the control or reference substance. KLH (Keyhole Limpet Hemocyanin) was used as the control or reference substance. The number of proliferating Th cells per well, or the number of CD3-positive, CD4-positive, and EdU-positive cells, was evaluated as the signal response. Based on the calculated SI, donors with an SI > 2 were considered positive. Furthermore, to reduce the risk of false negatives, a statistical equivalence test was also performed, using the Distribution Free Resampling (DFR) method described by Moody et al.

[0105] The results are shown in Table 3 and Figure 3. The number of donors who were statistically immunogenic positive for NB007-01 was 2 out of 20, indicating lower immunogenicity compared to the control substance. Similarly, the number of donors who were statistically immunogenic positive for NB007-02 was 7 out of 20, indicating lower immunogenicity compared to the control substance. Similarly, the number of donors who were statistically immunogenic positive for NB007-03 was 9 out of 20, indicating lower immunogenicity compared to the control substance. In particular, NB007-01, which had improved CDRs, had unexpectedly improved immunogenicity compared to NB007-03, which had no CDR improvements.

[0106] [Table 3]

[0107] [Example 4] Evaluation of cell binding by flow cytometry Binding to hCCR7 transfected cells and MJ cells, a human lymphoma cell line derived from T cell lymphoma, was assessed using NB007-01 labeled with the fluorescent dye Allophycocyanin (APC). Binding to Granta-519 cells, a human lymphoma cell line derived from B cell lymphoma, was assessed using unlabeled NB007-01 with an Alexa Fluor 647-conjugated anti-human IgG antibody (Thermo Fisher Scientific) as the secondary antibody. APC conjugation was performed using the APC Conjugation Kit - Lightning-Link (Abcam). 100 μL of antibody solution diluted to 1 mg / mL with PBS was added with 10 μL of Modifier Reagent, and the mixture was reacted with the APC Conjugation Mix. After storage at room temperature in the dark for 3 hours, 10 μL of Quencher Reagent was added and used as a fluorescently labeled antibody.

[0108] hCCR7 gene-transfected cells were detached from the culture dish using Cell Dissociation Buffer, enzyme-free, Hanks' Balanced Salt Solution (Thermo Fisher Scientific) and washed with PBS. Meanwhile, the required amount of suspension cells, Granta-519 cells and MJ cells, were each aliquoted and washed with PBS. 1 × 10 cells were added to PBS. 7 An equal volume of goat serum (Thermo Fisher Scientific) was added to the cell suspension, which was suspended at 100 μg / mL, and the mixture was left to stand at 4°C for 30 minutes for blocking. The mixture was centrifuged at 200 g for 5 minutes, the supernatant was removed, and 4 × 10 cells were collected. 6 Cells were suspended in FACS buffer (PBS containing 1% FBS) to a concentration of 1 / mL and dispensed in 50 μL aliquots into a 96-well plate. Fluorescently labeled NB007-01 antibody or unlabeled NB007-01 diluted in FACS buffer was mixed and incubated at 4°C for 1 hour. Cells were collected by centrifugation at 200g for 5 minutes and washed with 100 μL of FACS buffer. This washing procedure was repeated twice. hCCR7 transfected cells and MJ cells exposed to labeled NB007-01 were suspended in 100 μL of FACS buffer, and binding of the labeled antibody to the cells was measured using a CytoFLEX flow cytometer (Beckman Coulter). Granta-519 cells exposed to unlabeled NB007-01 were suspended in 50 μL of Alexa Fluor 647-labeled anti-human IgG antibody diluted 800-fold in FACS buffer and incubated at 4°C for 1 hour. The cells were washed twice and then suspended in 100 μL of FACS buffer, and antibody binding was measured using a flow cytometer.

[0109] Graphs plotting the antibody concentration on the horizontal axis and the geometric mean of the fluorescence intensity of the FACS histogram on the vertical axis are shown in Figures 4A to 4C. These graphs confirmed the specific binding of NB007-01 to hCCR7 transfected cells (Figure 4A), Granta-519 cells (Figure 4B), and MJ cells (Figure 4C).

[0110] [Example 5] Intracellular Ca 2+Functional evaluation of NB007-01 by signal measurement The hCCR7 gene-transfected cells were seeded in a 96-well microplate and cultured for 2 days in the same manner as in Example 2. The culture medium was replaced with a solution containing 3 μM Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Invitrogen). After 1 hour, 10 cells were added to each well. -6 ~10 -10 NB007-01 was added in a range of concentrations from 10 to 50 M. After 15 min, 5 × 10 -8 M CCL21 (R&D Systems) or 1.5 x 10 -8 Each cell line was stimulated with CCL19 (R&D Systems) at 1000 M. 2+ Using a signal measurement device, intracellular Ca 2+ The inhibitory activity of NB007-01 on intracellular Ca signaling was analyzed. The results are shown in Figure 5A and Figure 5B. 2+ 5A and 5B show graphs showing the relationship between inhibitory activity against signals and antibody concentration, with Fig. 5A representing the case where stimulation was with CCL21 and Fig. 5B representing the case where stimulation was with CCL19. Inhibitory activity was calculated as a relative value by standardizing the inhibition rate from "no antibody, but with CCL21 or CCL19" to 0% and "no antibody, but without CCL21 or CCL19" to 100%.

[0111] As shown in Figures 5A and 5B, NB007-01 inhibited CCL21- and CCL19-induced intracellular Ca 2+ The IC50 values ​​were 17.8 nM for CCL21 stimulation and 21.4 nM for CCL19 stimulation. These results suggest that NB007-01 inhibits CCL21- and CCL19-induced intracellular Ca signaling. 2+ It was shown to inhibit signaling.

[0112] [Example 6] Intracellular Ca using human lymphoma cell lines 2+ Signal transduction inhibitory activity evaluation MJ cells were washed with HBSS (containing CaCl2 and MgCl2) + 0.1% BSA assay buffer and 1.16 × 106 The cells were mixed at a cell concentration of 1 × 10 cells / mL with a solution containing 1 μM Cal-520 (AAT Bioquest) and 0.05% Pluronic-F127, and incubated at 37°C. After 45 minutes, the cells were washed twice with assay buffer and diluted to 1 × 10 cells / mL. 6 The cell concentration was adjusted to 1 / mL, and 80 μL was seeded into each well of a 96-well black PDL-coated microplate (CELLCOAT, Grenier). NB007-01 or CAP-100 was added to each well at 8 concentrations, 3-fold serial dilutions ranging from 250 nM to 0.11 nM, and the plate was incubated for 15 minutes. 2+ The cells were placed in a signal analyzer (FDSSμCELL; Hamamatsu Photonics) and stimulated with 20 nM CCL21 (R&D Systems) to measure intracellular Ca 2+ The inhibitory activity of each antibody against the signal was analyzed. The results for NB007-01 are shown in Figure 6A, and the results for CAP-100 are shown in Figure 6B. As shown in Figure 6A, NB007-01 inhibited CCL21-induced intracellular Ca signaling. 2+ CAP-100 inhibited CCL21-induced intracellular Ca signaling in a concentration-dependent manner, with a median inhibitory concentration (IC50) of 14.8 nM. On the other hand, as shown in Figure 6B, CAP-100 inhibited CCL21-induced intracellular Ca signaling. 2+ Although signal transduction was inhibited in a concentration-dependent manner, the maximum inhibition rate was approximately 50%.

[0113] [Example 7] Evaluation of cell migration inhibitory activity using human lymphoma cell lines Granta-519 cells were stained with CytoRed (Dojindo Laboratories) and then washed twice with RPMI + 0.5% BSA. 6 NB007-01 (0.3 μg to 100 μg / mL) or a negative control human IgG1 antibody (Icosagen) (1 μg / mL or 100 μg / mL) was added to a cell suspension at 100 cells / mL, and the mixture was left to stand at 37°C for 30 minutes.

[0114] 50 ng / mL CCL19 was added to the wells of a 96-well Transwell receiver plate with an integrated insert and a 3.0 μm polycarbonate membrane (Corning). After placing the Transwell insert on the receiver plate, 50 μL of the cell suspension was seeded into the insert and incubated at 37°C. After incubation for 4 hours, the insert was removed, and each well was photographed in three fields using a 4x fluorescent lens to count the number of migrated cells. The inhibitory activity was calculated as a relative value, normalized to 0% inhibition for "no antibody, with CCL19" and 100% inhibition for "no antibody, without CCL19." Figure 7 shows the inhibitory effect of NB007-01 on CCL19-dependent cell migration. The IC50 of NB007-01 was 25.5 nM. The inhibition rates of the negative control human IgG1 antibody at 1 μg / mL and 100 μg / mL were 3.0% and 3.3%, respectively.

[0115] [Example 8] Preparation of defucosylated NB007-01 The antibody was defucosylated using the GlymaxX method (WO 2011 / 035884, Japanese Patent No. 5746183). Following the procedure used to produce NB007-01 using the QMCF method in Example 1, a CHO cell line (CHOEBNALT85-RMD C4) modified by the GlymaxX method was used as the CHO cells for antibody gene expression to produce defucosylated NB007-01. The antibody was purified.

[0116] N-glycans were removed from purified antibodies and labeled using the AdvanceBio Gly-XN-glycan prep with InstantPC Kit (Agilent) and sialidase (AdvanceBio Sialidase A) (Agilent, GK80040), β1-3,4 Galactosidase (β1-3,4 Galactosidase; BTG) (NEB, P0746S), β-N-Acetylglucosaminidase S (GUH) (NEB, P0744), and α1-2,4,6 Fucosidase O (FucO) (NEB, P0749) according to the manufacturer's instructions. The labeled N-glycans were analyzed by HPLC (Agilent 1260 Infinity II) using an AdvanceBio Glycan 2.7 μm column (Agilent) according to the manufacturer's instructions. Figure 8A shows the HPLC chromatogram, and Figure 8B shows the abundance of each glycan species calculated from the chromatogram. In Figure 8A, "Undig" indicates untreated, "Sialidase A" indicates sialidase treatment, "Sial+BTG" indicates sialidase + BTG treatment, "Sial+BTG+GUN" indicates sialidase + BTG + GUN treatment, and "Sial+BTG+GUN+FucO" indicates sialidase + BTG + GUN + FucO treatment. As shown in Figures 8A and 8B, the majority of the resolved peaks were assigned to specific glycans based on their relative retention times (glucose units), accounting for 89% of the peak area for all samples. Furthermore, in the cleavage experiment with FucO, there was almost no change in the peaks, and more than 99% of the identified glycan species were defucosylated.

[0117] Example 9: Evaluation of the cytotoxic function of defucosylated NB007-01 (1) ADCC reporter bioassay The ADCC function of the defucosylated antibodies was evaluated using the ADCC Reporter Bioassay, V Variant (Promega). 1.4 mL of Low IgG Serum (included in the kit) was mixed with 33.6 mL of RPMI 1640 Medium to prepare the ADCC Assay Buffer. 5 × 10 Granta-519 cells were cultured in the buffer. 5 The cells were suspended to a cell density of 1000 / mL. Additionally, a diluted solution of the antibody test substance was prepared using ADCC Assay Buffer. The test substances used were NB007-01, defucosylated NB007-01, CAP-100, and a negative control human IgG (Icosagen). A 630 μL aliquot of the ADCC Bioassay Effector Cells provided with the kit was added to 3.6 mL of ADCC Assay Buffer to prepare an effector cell suspension. 25 μL each of the antibody dilution, Granta-519 cell suspension, and effector cell suspension was dispensed into a Culture Plate-96 (PerkinElmer) and incubated at 37°C in a 5% CO2 environment for 6 hours. The Luciferase Assay Substrate provided with the kit was dissolved in Luciferase Assay Buffer, and 75 μL was added to each well of the plate. After 15 minutes of incubation in the dark at room temperature, the relative luminescence units (RLU) were measured using an ARVO microplate reader (PerkinElmer). Figure 9 shows a graph plotting antibody concentration on the horizontal axis and RLU on the vertical axis. RLU is an indicator of effector cell activation, and this bioassay is a functional measurement method based on the ADCC mechanism of action. As shown in Figure 9, defucosylated NB007-01 (EC50 = 16.1 pM) exhibited significantly improved ADCC function compared to NB007-01 (EC50 = 123 pM) and superior to CAP-100 (EC50 = 57.1 pM).

[0118] (2) Cytotoxicity assay using human PBMCs (a) Preparation of human PBMCs Frozen human PBMCs were thawed in a water bath at 37°C. After washing with complete medium, 2 × 106 The cells were resuspended in complete medium to a concentration of 1 / mL and cultured overnight at 37°C under 5% CO2 conditions.

[0119] (b) Pretreatment of Granta-519 cells Granta-519 cells were cultured in ADCC medium at a viable cell concentration of 3 × 10 4 The solution was prepared at a volume of 80 μL per well and seeded into each well of a 96-well round-bottom plate. 20 μL of test substance diluted in ADCC medium was then added to each well and allowed to stand at room temperature for 30 minutes. The test substances used were defucosylated NB007-01 (1000-0.0001 ng / mL), CAP-100 (10,000-0.1 ng / mL), and Rituximab (10,000 ng / mL, Roche).

[0120] (c) Measurement of ADCC activity Human PBMCs were used as effector cells (E) and Granta-519 cells as target cells (T) at an E:T ratio of 50:1. Specifically, the human PBMCs (a) were washed with ADCC medium, and the cell concentration was adjusted to 1.5 × 10 6 100 μL of cells were added to each well of (b). After incubation at 37°C for 5 hours, the cells were centrifuged at 250 × g for 4 minutes, and 50 μL of the supernatant was collected into each well of a clear flat-bottom 96-well plate. Forty-five minutes before collecting the supernatant, 20 μL of lysis solution (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) was added to the wells to provide a control for maximum cytotoxicity.

[0121] 50 μL of substrate mixture (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) was added to each well, and the plate was mixed for 30 seconds. The plate was then left to stand at room temperature for 30 minutes. 50 μL of stop reagent (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) was added to each well, mixed for 30 minutes, and absorbance was measured at 490 nm. Cytotoxic activity (%) was calculated as a relative value, normalized to 100% for maximum cytotoxicity and 0% for no test substance. A graph plotting the calculated values ​​is shown in Figure 10. In Figure 10, "aNB007" refers to defucosylated NB007-01.

[0122] The results of this study also showed that defucosylated NB007-01 (EC50 = 1.9 ng / mL, 12.6 pM in IgG equivalent) demonstrated superior cytotoxic activity to CAP-100 (EC50 = 26.3 ng / mL, 175 pM in IgG equivalent). Furthermore, Rituximab, which is clinically used for its antitumor activity based on ADCC activity, only achieved 18.6% cytotoxicity at a concentration of 10,000 ng / mL, suggesting the superiority of defucosylated NB007-01 in tumor therapeutic efficacy.

[0123] [Example 10] Antitumor activity of humanized anti-CCR7 antibodies The antitumor activity of NB007-01, defucosylated NB007-01, and CAP-100 was evaluated in a tumor xenograft model using Granta-519 cells. Female CB17 / SCID mice (6-8 weeks old) were used as the model animals.

[0124] Granta-519 cells were cultured in RPMI-1640 medium containing 20% ​​FBS at 37°C under 5% CO2. 7Granta-519 cells were transplanted intravenously into 6-8 week-old CB17 / SCID mice at a concentration of 0.1 mL per cell. The day of transplantation was designated as day 0. On day 1 after transplantation, mice were divided into groups based on their body weight. Group division was performed using the matched distribution method (StudyDirectory). TM This was performed using the NIRS software, version 3.1.399.19.

[0125] Administration of the test substances began two days after cancer cell transplantation. The dose was 3 mg / kg for NB007-01, CAP-10, and the isotype control human IgG1, and 0.03 mg / kg for defucosylated NB007-01. Each test substance was administered in a volume of 10 μL / g twice a week.

[0126] After cancer cell transplantation, each animal was monitored daily for morbidity and mortality. Additionally, any abnormalities in mobility, food and water consumption, weight gain or loss, and eye and coat condition due to tumor growth or treatment were monitored. Body weight was measured twice a week. Details of deaths and clinical signs were recorded for each individual. Weight was recorded by the Study Director. TM The antitumor activity was evaluated by Kaplan-Meier survival curves based on the survival rate of each individual.

[0127] In addition, livers and lymph nodes were harvested from some animals in each group 24 days after cancer cell transplantation. The harvested organs were fixed in 10% formalin buffer and paraffin blocks were prepared. The prepared blocks were cut into 4-μm sections and treated at 60°C for 30 minutes. The infiltration rate of Granta-519 cells was measured by immunostaining. Granta-519 cells were identified using an antibody against human CD45 (Cell Signaling Technology, Cat#13917), a hematopoietic cell surface marker. Sections were activated in EDTA, pH 9.0, at 100°C for 20 minutes. The antibody was diluted 1:800. All immunostained slides were scanned at 40x magnification using the NanoZoomer-HT 2.0 / Pannoramic SCAN Image system and saved. The number of CD45-positive cells was measured using the HALO™ platform for immunostaining scoring, and the number of cells was calculated by dividing the total by 1 mm. 2 The number of cells was evaluated per 100 cells.

[0128] Figure 11 shows the progression of survival rates as expressed by Kaplan-Meier survival curves. Figure 12A shows the number of CD45-positive cells in lymph nodes, and Figure 12B shows the number of CD45-positive cells in the liver. As shown in Figure 11, NB007-01 extended survival compared to the isotype control group and even extended survival more than CAP-100. As shown in Figures 12A and 12B, NB007-01 strongly inhibited cancer cell infiltration into various organs, and the effect was stronger than CAP-100 in the liver. This suggests that NB007-01 exerts its antitumor effect by inhibiting lymphoma infiltration into various organs. Furthermore, defucosylated NB007-01 showed a survival benefit comparable to that of CAP-100 at a dose 1 / 100 of that of NB007-01. These results suggest that defucosylated NB007-01, with its enhanced cytotoxic activity, may be effective at low doses.

Claims

1. An antibody that specifically binds to the extracellular domain of human CCR7, a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 27; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 29; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37; and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39; A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 21, and An antibody having a light chain variable region comprising the amino acid sequence represented by sequence number 31.

2. having an Fc region, wherein the Fc region comprises an N-glycoside-linked sugar chain; fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain; The antibody according to claim 1, which has antibody-dependent cellular cytotoxicity.

3. An antibody that specifically binds to the extracellular domain of human CCR7, a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 45; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 49; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 57; and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:59; A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 41, and An antibody having a light chain variable region comprising the amino acid sequence represented by sequence number 51.

4. having an Fc region, wherein the Fc region comprises an N-glycoside-linked sugar chain; fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain; The antibody according to claim 3, which has antibody-dependent cellular cytotoxicity.

5. A nucleic acid encoding the antibody of any one of claims 1 to 4.

6. The nucleic acid of claim 5, comprising a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 21 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:

31.

7. The nucleic acid of claim 5, comprising a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 41 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:

51.

8. A cell comprising the nucleic acid of claim 5.

9. A cell comprising the nucleic acid of claim 6.

10. A cell comprising the nucleic acid of claim 7.

11. A pharmaceutical comprising the antibody according to any one of claims 1 to 4 as an active ingredient.

12. The pharmaceutical composition according to claim 11, which is used for the treatment of cancer.

13. The pharmaceutical composition of claim 12, wherein the cancer is a blood cancer.

14. The pharmaceutical composition of claim 13, wherein the blood cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or non-Hodgkin's lymphoma.

15. The pharmaceutical composition of claim 14, wherein the non-Hodgkin's lymphoma is B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, chronic lymphocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, or cutaneous lymphoma.

16. The pharmaceutical composition of claim 12, wherein the cancer is a solid cancer.

17. The pharmaceutical composition of claim 16, wherein the solid cancer is breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, cervical cancer, hepatocellular carcinoma, urothelial carcinoma, renal cell carcinoma, or squamous cell carcinoma.

18. The pharmaceutical composition of claim 17, wherein the squamous cell cancer is oral squamous cell carcinoma, esophageal squamous cell carcinoma, or pharyngeal squamous cell carcinoma.

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